Moraxella catarrhalis can cause otitis media which is of significant public health and economic concern in humans. Vaccine strategies are being pursued to combat these infections. These strategies are based on the identification of conserved, immunogenic cell surface components; however, the detection of conserved molecules that would confer protection against the vast majority of strains from a single species has proven problematic.
The outer leaflet of the outer membrane of all Gram-negative bacteria contains an amphiphillic carbohydrate molecule termed lipopolysaccharide (LPS).
An O-antigenic polymeric repeating unit (O-antigen) can be present or absent beyond the core oligosaccharide of the LPS molecule and is absent in all strains of Moraxella catarrhalis so far examined. The core oligosaccharide can be arbitrarily divided into an outer and inner core and is connected to the lipid A region via one or more ketose sugar(s), 2-keto-3-deoxy-octulosonic acid (Kdo). The lipid A region is responsible for the endotoxic activity of the Gram-negative bacterium and consists in most species of a disaccharide of glucosamine sugars that are phosphorylated and contain both ester and amide linked fatty acids. The outer core region can be somewhat variable within a species and is therefore not a good vaccine candidate. However what is arbitrarily termed the inner core oligosaccharide has been found to be conserved within several species, and is the vaccine antigen of choice in this application.
The endotoxicity of the lipid A region is due to the fatty acid residues. Removal of the ester-linked fatty acids leaves an O-deacylated LPS species that is no longer endotoxic. Removal of all fatty acids i.e. both the amide and ester-linked fatty acids can be performed chemically, but involves harsh conditions which can sometimes affect other regions of the LPS molecule if residues susceptible to these conditions are elaborated by the bacterial species LPS of interest. Therefore if a conserved residue is removed by the conditions employed to prepare the vaccine antigen, it is likely that the resulting immune response to that antigen would not be broadly cross reactive or protective. We have previously detailed the utilisation of amidases from Dictyostelium discoideum to remove the N-linked fatty acids and thus avoid the use of harsh chemical conditions (and the possible removal of sensitive residues).
LPS based vaccines generally require the removal of sufficient fatty acids from the lipid A region of the molecule to preclude endotoxicity and to derive a molecule that is amenable to conjugation strategies.
Current strategies used in the art to prepare LPS-based glycoconjugate vaccines link the carbohydrate to a carrier protein either via the Kdo residues of O-deacylated LPS or of core oligosaccharides or via the derived lipid A region of the molecule. We have shown previously that conjugation via the Kdo residues does not optimally present the target core oligosaccharide region to the host's immune system and the resulting sera are not functional.